This invention relates to substances that inhibit neovascularization and to methods for their production and use.
Neovascularization plays a crucial role in the pathogenesis of several important human disorders, including diabetic retinopathy, senile macular degeneration, tumor growth, rheumatoid arthritis, and excessive scarring during wound healing. In diabetic retinopathy, new blood vessels grow from the optic disc and retina, which eventually can cause blindness. Laser treatment has been shown to reduce blindness from this disorder, but the destruction caused by the formation of retinal scars causes dramatic reductions in peripheral vision and night vision. Pharmacologic inhibitors of neovascularization could provide improved treatment modalities for patients with this disease.
In senile macular degeneration, new blood vessels grow through Bruch's membrane to invade the retina. This retinal invasion causes destruction of the photoreceptors and thereby reduces vision. Inhibitors of neovascularization could prevent or limit loss of vision.
The process of tumor growth and invasion is one of the major causes of mortality in the industrialized nations. The inhibition of neovascularization has been shown to induce the regression of tumors. Availability of therapeutic quantities of a potent inhibitor of neovascularization could provide an important adjunct to current cancer therapy.
In rheumatoid arthritis, the articular cartilage of the involved joint is invaded by a vascular pannus. This vascular tissue destroys the normal smooth cartilage surface. Inhibition of neovascularization could lessen the joint destruction that occurs in rheumatoid arthritis.
Excessive scarring during wound healing, such as in the case of keloids, may cause significant disfiguration. Since neovascularization is an important component of wound healing and scar formation, its inhibition may control keloid formation.
The possibility that controlling neovascularization will aid in the treatment of these disorders has prompted an extensive search for inhibitors of new blood vessel formation. Most inhibitors of neovascularization so far identified have been extracted from tissues that are avascular, such as cartilage, vitreous, and lens. For example, Jacobson et al. found a low molecular weight (less than 13,000 daltons) substance or substances that inhibited aortic endotheolial cell proliferation in several isolates that had been derived from human vitreous. The isolates had been physically extracted from the vitreous, centrifuged, and subjected to gel chromatography. Jacobson et al., Arch. Ophthalmol., 102, 1543 (1984). Williams et al. described a substance with a molecular weight of less than 100,000 that inhibited bovine aortic endothelial cell proliferation. The substance was extracted from human and bovine lenses by 1 M quanidine hydrochloride and was passed through a membrane with a molecular weight cut-off of 100,000 daltons. Williams, et al., Am. J. Ophthalmol., 97, 366 (1984). Lutty et al. described an extract from adult bovine vitreous that inhibited neovascularization. The extract was prepared by homogenizing the vitreous, incubating it with sodium ascorbate, dialyzing it (12,000-14,000 molecular weight cut-off), and filter-sterilizing the dialyzate. Lutty et al., Investigative Ophthalmology and Visual Science, 23, 52 (1983). Brem et al. found a factor extracted from rabbit vitreous that inhibited the growth of new blood vessels induced by tumors in rabbit corneas. The factor was extracted by centrifuging vitreous and dialyzing the supernatant, using cellulose tubing with a 12,000 molecular weight limit. Brem et al., Am. J. Ophthalmol., 84, 323 (1977).
Unfortunately, these substances presently are not useful for the treatment of diseases involving neovascularization. Only limited quantities of inhibitors can be extracted from the previously mentioned sources, and the inhibitors have been only partially purified and characterized. In fact, the limited quantities and relatively unpurified state of these substances have made it difficult even to evaluate their potential for use in the treatment of disease.
U.S. Pat. No. 4,356,261 to Kuettner discloses a process for producing a neovascularization inhibitor derived from cartilage, which is intended to overcome the problem of the limited supply of such substances. The method involves culturing cartilage producing cells at high density and extracting the inhibitor from the culture. The extraction process is disclosed in U.S. Pat. No. 4,042,457 to Kuettner et al. The process involves the use of an aqueous extraction medium that includes a solute that does not irreversibly denature the proteinaceous material to be extracted, preferably a 1.0-3.0 M aqueous solution of quanidine hydrochloride, separating the extract, recovering substances having a molecular weight below about 50,000, treating such substances to remove salts therefrom, and dehydrating the resultant material. The substance or substances, which has a molecular weight of 50,000 or less, inhibits the rate of proliferation of endothelial cells. It is only partially purified and characterized and, therefore, is not useful for the treatment of the above-mentioned disorders.
Intraocular neovascularization occurring in diabetic retinopathy is unique in that a therapy exists whereby regression of new blood vessels can be induced and future neovascularization inhibited. This therapy is based on the observation that diabetic intraocular neovascularization rarely occurs in eyes with chorioretinal scars. This has led to the widespread use of argon laser and xenon photocoagulation to therapeutically induce chorioretinal scar formation. The production of photocoagulation induced chorioretinal scars results in a rapid regression of intraocular neovascularization in eyes with proliferative diabetic retinopathy. Such regression occurs even when photocoagulation and resultant chorioretinal scarring occurs in areas remote from the new blood vessels.
Numerous theories have been proposed to explain this phenomenon, but none have as yet been substantiated. One theory suggests that photocoagulation increases the amount of oxygen released into the eye, which is thought to inhibit neovascularization. However, this sequence of events is unproven. Stefansson, et al., Ophthalmic Surgery, 14, 209 (1983). Another theory is that photocoagulation destroys retina that releases a stimulus for neovascularization, but this also is unproven. The main reason for doubting the theory is the fact that photocoagulation does not uniformly destroy the inner aspects of the retina where the stimulator for neovascularization is thought to be produced. A third theory is that the photocoagulation allows an escape route for the stimulators of neovascularization to leave the eye. Foulds, Trans. Ophthalmol. Soc. NZ, 32, 82 (1980). There is no proof to support this theory either.
The inventor has discovered that cellular components of chorioretinal scars release a substance that inhibits neovascularization and, more specifically, that such inhibitor is released by particular types of cells found in the scars. Chorioretinal scars are composed mainly of astrocytes, retinal pigment epithelial cells, and possibly fibroblasts. Upon testing the ability of these three types of cells to release a substance that causes the regression of new blood vessels in vitro, the present inventor has found that certain retinal pigment epithelial cells and fibroblast cells in culture release such a substance. The substance has been isolated, purified, and characterized.
This discovery is quite unexpected in view of the different theories purporting to explain the effects of chorioretinal scarring and the fact that some recent articles would steer a person skilled in the art away from looking at retinal pigment epithelial cells as a source of neovascularization inhibitor. For example, Korte et al. and Heriot et al. have suggested that retinal pigment epithelial cells may release a substance necessary for blood vessel maintenance. Korte et al., Invest. Ophthalmol. Vis. Sci., 25, 1135 (1984). Heriot et al., Opthalmology, 91, 1603 (1984).
The neovascularization inhibitors of the present invention are produced by cultured cells, including retinal pigment epithelial cells of humans and certain other animals and human fibroblast cells. This provides major advantages over the method of producing neovascularization inhibitors from cartilage, vitreous, and lens by extraction. First, the yield of inhibitor is significantly greater than for the extraction process because very large quantities of inhibitor can be produced by using techniques well-known in the art for mass cell culture. The production of sufficient quantities of neovascularization inhibitor permits its isolation, purification, and characterization for further study and also permits sufficient production for commercial applications, such as the treatment of the previously mentioned disorders. Second, the ability to culture cells that produce the inhibitor provides the opportunity to isolate the DNA coding for the active substance, introduce the DNA into bacteria or other organisms, and achieve large-scale synthesis of the active molecule. The employment of such genetic engineering techniques should allow the production of neovascularization inhibitors more cheaply than with mass cell culture techniques and would allow the modification of the molecule to produce analogs with possibly enhanced activity.